PROJECT SUMMARY/ABSTRACT The initiation and propagation of an immune response are dependent on integration of pathogen- and host-derived signals which shape and amplify immunity.1 The right combination of these signals results in robust proliferation of T cells that acquire effector functions like cytokine production and cytotoxicity. How T cells support this robust proliferation and subsequent effector function, from a metabolic perspective, has garnered much interest, and it is now clear that metabolic and nutrient sensing pathways represent key mechanisms by which T cells are regulated.2 Glucose is utilized as a primary fuel and its processing via aerobic glycolysis is not only required for proliferation of effector T cells, but is also necessary for post- transcriptional stability of effector gene transcripts.3 Mitochondria serve as generators of biomass, promote longevity for nave and memory T cells, produce ATP when glucose is limiting, buffer calcium, and generate ROS required for cytokine production.4, 5 Importantly, these pathways can be deregulated, resulting in exuberant T cell responses in autoimmunity or T cell dysfunction in cancer.6 However, while most studies have been focused on the genetic and pharmacologic blockade of these entire pathways and ascertaining relatively long-term effects on T cell function and fate, it is clear from work done in other cellular systems that energetic changes occur extremely rapidly. Furthermore, studies using microscopic techniques have revealed that T cell activation is neither binary nor ubiquitous in the cell, but occurs in a graded fashion at the immunologic synapse (IS).7 Thus, it is still unclear how metabolism and T cell activation intersect spatially, within the cell, to enable effector function. Our laboratory studies how T cell metabolism is regulated both during normal activation and in pathologic conditions such as within the tumor microenvironment. In this application, we focus on the spatiotemporal regulation of bioenergetics using an innovative set of new technologies to study metabolism using microscopic techniques. We hypothesize that early metabolic changes induced by TCR ligation license effector function originating at the immunologic synapse. Aim 1: Identify how immediate-early glycolysis regulates effector function at the immunologic synapse Our exciting preliminary data have revealed that aerobic glycolysis occurs immediately after T cell activation, in response to TCR ligation alone, through the activation of the pyruvate dehydrogenase kinase 1 (PDHK1). Further, we have found a central role for lactate dehydrogenase, which catalyzes the glycolytic conversion of pyruvate to lactate, as an RNA binding protein controlling effector gene transcripts. We hypothesize glycolysis occurs locally at the IS to enable translation of specific effector genes in response to TCR activation. 1A) Visualize early activation-induced glycolysis at the single cell level. Using cutting-edge systems to study glycolysis at the immunologic synapse, we will ascertain the dynamics of glycolysis induced in response to TCR ligation and other stimuli, and whether cytokine translation is differentially regulated at that site. 1B) Determine whether glycolysis enables cytokine synthesis at the IS. T cells orient cytokine production toward the immunologic synapse. Using microscopy techniques, we will determine whether cytokine translation at the immunologic synapse is associated with TCR-elicited glycolysis. Aim 2: Elucidate the local role of mitochondria as mediators of T cell effector function We have previously shown that tumor-infiltrating T cells lose mitochondrial mass and that this `metabolic exhaustion' underlies their dysfunction in the tumor microenvironment.8 However, it is still unclear by what mechanism mitochondria are required in the acquisition of effector function and the avoidance of dysfunction. We hypothesize that mitochondria are required to locally fuel effector function and induce sustained calcium flux in T cells, a process that will be deranged in exhausted T cells lacking mitochondria. 2A) Measure early-activation-induced mitochondrial dynamics in distinct T cell functional states . Using a novel technology that allows for high resolution mitochondrial imaging, we will determine the mitochondrial dynamics during early activation of T cells `conditioned' in vivo to generate mitochondria-rich memory cells or mitochondria-poor exhausted cells. 2B) Determine the requirement for mitochondrial activity in T cell activation. The same imaging platform utilized to image mitochondria can also be employed to acutely photoablate mitochondrial activity, allowing for non-pharmacologic, optogenetic methods to determine the role for mitochondria in T cell activation. We will utilize this system to determine how mitochondria may be required to promote calcium flux/NFAT signaling, generate ATP, or influence glycolysis at the immunologic synapse. We believe these studies will provide fundamental insight into how T cell effector function is regulated by metabolism by understanding how these pathways interact at the spatiotemporal level. Importantly, as metabolic means are being considered for immunomodulatory therapies, it is thus important to understand the requirement of these interactions for survival and effector function of T cells in vivo.